Inositol Polyphosphate Multikinase Inhibits Angiogenesis via Inositol Pentakisphosphate-Induced HIF-1? Degradation.
ABSTRACT: RATIONALE:Inositol polyphosphate multikinase (IPMK) and its major product inositol pentakisphosphate (IP5) regulate a variety of cellular functions, but their role in vascular biology remains unexplored. OBJECTIVE:We have investigated the role of IPMK in regulating angiogenesis. METHODS AND RESULTS:Deletion of IPMK in fibroblasts induces angiogenesis in both in vitro and in vivo models. IPMK deletion elicits a substantial increase of VEGF (vascular endothelial growth factor), which mediates the regulation of angiogenesis by IPMK. The regulation of VEGF by IPMK requires its catalytic activity. IPMK is predominantly nuclear and regulates gene transcription. However, IPMK does not apparently serve as a transcription factor for VEGF. HIF (hypoxia-inducible factor)-1? is a major determinant of angiogenesis and induces VEGF transcription. IPMK deletion elicits a major enrichment of HIF-1? protein and thus VEGF. HIF-1? is constitutively ubiquitinated by pVHL (von Hippel-Lindau protein) followed by proteasomal degradation under normal conditions. However, HIF-1? is not recognized and ubiquitinated by pVHL in IPMK KO (knockout) cells. IP5 reinstates the interaction of HIF-1? and pVHL. HIF-1? prolyl hydroxylation, which is prerequisite for pVHL recognition, is interrupted in IPMK-deleted cells. IP5 promotes HIF-1? prolyl hydroxylation and thus pVHL-dependent degradation of HIF-1?. Deletion of IPMK in mouse brain increases HIF-1?/VEGF levels and vascularization. The increased VEGF in IPMK KO disrupts blood-brain barrier and enhances brain blood vessel permeability. CONCLUSIONS:IPMK, via its product IP5, negatively regulates angiogenesis by inhibiting VEGF expression. IP5 acts by enhancing HIF-1? hydroxylation and thus pVHL-dependent degradation of HIF-1?.
Project description:The hypoxia-inducible factor-1alpha (HIF-1alpha) pathway is the central regulator of adaptive responses to low oxygen availability and is required for normal skeletal development. Here, we demonstrate that the HIF-1alpha pathway is activated during bone repair and can be manipulated genetically and pharmacologically to improve skeletal healing. Mice lacking pVHL in osteoblasts with constitutive HIF-1alpha activation in osteoblasts had markedly increased vascularity and produced more bone in response to distraction osteogenesis, whereas mice lacking HIF-1alpha in osteoblasts had impaired angiogenesis and bone healing. The increased vascularity and bone regeneration in the pVHL mutants were VEGF dependent and eliminated by concomitant administration of VEGF receptor antibodies. Small-molecule inhibitors of HIF prolyl hydroxylation stabilized HIF/VEGF production and increased angiogenesis in vitro. One of these molecules (DFO) administered in vivo into the distraction gap increased angiogenesis and markedly improved bone regeneration. These results identify the HIF-1alpha pathway as a critical mediator of neoangiogenesis required for skeletal regeneration and suggest the application of HIF activators as therapies to improve bone healing.
Project description:Hypoxia-inducible factor (HIF)-1alpha is a short-lived protein and is ubiquitinated and degraded through the von Hippel-Lindau protein (pVHL)-E3 ubiquitin ligase pathway at normoxia. Deubiquitination, by reversing ubiquitination, has been recognized as an important regulatory step in ubiquitination-related processes. Here, we show that pVHL-interacting deubiquitinating enzyme 2, VDU2, but not VDU1, interacts with HIF-1alpha. VDU2 can specifically deubiquitinate and stabilize HIF-1alpha and, therefore, increase expression of HIF-1alpha targeted genes, such as vascular endothelial growth factor (VEGF). These findings suggest that ubiquitination of HIF-1alpha is a dynamic process and that ubiquitinated HIF-1alpha might be rescued from degradation by VDU2 through deubiquitination. Although pVHL functions as a master control for HIF-1alpha stabilization, as pVHL-E3 ligase mediates the ubiquitination of both HIF-1alpha and VDU2, the balance between the pVHL-mediated ubiquitination and VDU2-mediated deubiquitination of HIF-1alpha provides another level of control for HIF-1alpha stabilization.
Project description:Gag polymerization with viral RNA at the plasma membrane initiates HIV-1 assembly. Assembly processes are inefficient in vitro but are stimulated by inositol (1,3,4,5,6) pentakisphosphate (IP5) and inositol hexakisphosphate (IP6) metabolites. Previous studies have shown that depletion of these inositol phosphate species from HEK293T cells reduced HIV-1 particle production but did not alter the infectivity of the resulting progeny virions. Moreover, HIV-1 substitutions bearing Gag/CA mutations ablating IP6 binding are noninfectious with destabilized viral cores. In this study, we analyzed the effects of cellular depletion of IP5 and IP6 on HIV-1 replication in T cells in which we disrupted the genes encoding the kinases required for IP6 generation, IP5 2-kinase (IPPK) and Inositol Polyphosphate Multikinase (IPMK). Knockout (KO) of IPPK from CEM and MT-4 cells depleted cellular IP6 in both T cell lines, and IPMK disruption reduced the levels of both IP5 and IP6. In the KO lines, HIV-1 spread was delayed relative to parental wild-type (WT) cells and was rescued by complementation. Virus release was decreased in all IPPK or IPMK KO lines relative to WT cells. Infected IPMK KO cells exhibited elevated levels of intracellular Gag protein, indicative of impaired particle assembly. IPMK KO compromised virus production to a greater extent than IPPK KO suggesting that IP5 promotes HIV-1 particle assembly in IPPK KO cells. HIV-1 particles released from infected IPPK or IPMK KO cells were less infectious than those from WT cells. These viruses exhibited partially cleaved Gag proteins, decreased virion-associated p24, and higher frequencies of aberrant particles, indicative of a maturation defect. Our data demonstrate that IP6 enhances the quantity and quality of virions produced from T cells, thereby preventing defects in HIV-1 replication.
Project description:TLX is known as the orphan nuclear receptor indispensable for maintaining neural stem cells in adult neurogenesis. We report here that neuroblastoma cell lines express high levels of TLX, which further increase in hypoxia to enhance the angiogenic capacity of these cells. The proangiogenetic activity of TLX appears to be induced by its direct binding to the von Hippel-Lindau protein (pVHL), which stabilizes TLX. In turn, TLX competes with hydroxylated hypoxia-inducible factor (HIF-?) for binding to pVHL, which contributes to the stabilization of HIF-2? in neuroblastoma during normoxia. Upon hypoxia, TLX increases in the nucleus where it binds in close proximity of the HIF-response element on the VEGF-promoter chromatin, and, together with HIF-2?, recruits RNA polymerase II to induce VEGF expression. Conversely, depletion of TLX by shRNA decreases the expression of HIF-2? and VEGF as well as the growth-promoting and colony-forming capacity of the neuroblastoma cell lines IMR-32 and SH-SY5Y. On the contrary, silencing HIF-2? will slightly increase TLX, suggesting that TLX acts to maintain a hypoxic environment when HIF-2? is decreasing. Our results demonstrate TLX to play a key role in controlling angiogenesis by regulating HIF-2?. TLX and pVHL might counterbalance each other in important fate decisions such as self-renewal and differentiation, as well as angiogenesis and anti-angiogenesis.
Project description:The inositol polyphosphates are a group of multifunctional signaling metabolites whose synthesis is catalyzed by a family of inositol kinases that are evolutionarily conserved from yeast to humans. Inositol polyphosphate multikinase (IPMK) was first identified as a subunit of the arginine-responsive transcription complex in budding yeast. In addition to its role in the production of inositol tetrakis- and pentakisphosphates (IP4 and IP5), IPMK also exhibits phosphatidylinositol 3-kinase (PI3-kinase) activity. Through its PI3-kinase activity, IPMK activates Akt/PKB and its downstream signaling pathways. IPMK also regulates several protein targets non-catalytically via protein-protein interactions. These non-catalytic targets include cytosolic signaling factors and transcription factors in the nucleus. In this review, we highlight the many known functions of mammalian IPMK in controlling cellular signaling networks and discuss future challenges related to clarifying the unknown roles IPMK plays in physiology and disease.
Project description:The hypoxia-inducible factor-1? (HIF-1?) plays a critical role in tumor angiogenesis. It has been reported that the acetone extract of Angelica sinensis (AE-AS) rich in phthalides is able to inhibit cancer cell proliferation. However, whether AE-AS reduces cancer angiogenesis remains unknown. In this study, we demonstrated that AE-AS significantly inhibited the angiogenesis in vitro and in vivo evidenced by attenuation of the tube formation in hypoxic human umbilical vascular endothelial cells (HUVECs), and the vasculature generation in Matrigel plug, the chicken chorioallantoic membrane, and tumors. Treatment with AE-AS markedly decreased the protein accumulation and transcriptional activity of HIF-1?, vascular endothelial growth factor (VEGF) expression/secretion, and VEGFR2 phosphorylation in hypoxic human bladder cancer (T24) cells and tumor tissues accompanied by a reduction of tumor growth. Notably, AE-AS-induced HIF-1? protein degradation may, at least partly, attribute to inhibition of WSB-1-dependent pVHL degradation. Moreover, VEGFR2-activated PI3K/AKT/mTOR signaling pathway in hypoxic T24 cells was greatly inhibited by AE-AS. Collectively, AE-AS may be a potential anticancer agent by attenuating cancer angiogenesis via suppression of WSB-1/pVHL/HIF-1?/VEGF/VEGFR2 cascade.
Project description:Inositol polyphosphate multikinase (IPMK) is required for the biosynthesis of inositol phosphates (IPs) through the phosphorylation of multiple IP metabolites such as IP3 and IP4. The biological significance of IPMK's catalytic actions to regulate cellular signaling events such as growth and metabolism has been studied extensively. However, pharmacological reagents that inhibit IPMK have not yet been identified. We employed a structure-based virtual screening of publicly available U.S. Food and Drug Administration-approved drugs and chemicals that identified the antidepressant, vilazodone, as an IPMK inhibitor. Docking simulations and pharmacophore analyses showed that vilazodone has a higher affinity for the ATP-binding catalytic region of IPMK than ATP and we validated that vilazodone inhibits IPMK's IP kinase activities in vitro . The incubation of vilazodone with NIH3T3-L1 fibroblasts reduced cellular levels of IP5 and other highly phosphorylated IPs without influencing IP4 levels. We further found decreased Akt phosphorylation in vilazodone-treated HCT116 cancer cells. These data clearly indicate selective cellular actions of vilazodone against IPMK-dependent catalytic steps in IP metabolism and Akt activation. Collectively, our data demonstrate vilazodone as a method to inhibit cellular IPMK, providing a valuable pharmacological agent to study and target the biological and pathological processes governed by IPMK.
Project description:Activating transcription factor 4 (ATF4) is a critical transcription factor for bone remodeling; however, its role in bone angiogenesis has not been established. Here we show that ablation of the Atf4 gene expression in mice severely impaired skeletal vasculature and reduced microvascular density of the bone associated with dramatically decreased expression of hypoxia-inducible factor 1? (HIF-1?) and vascular endothelial growth factor (VEGF) in osteoblasts located on bone surfaces. Results from in vivo studies revealed that hypoxia/reoxygenation induction of HIF-1? and VEGF expression leading to bone angiogenesis, a key adaptive response to hypoxic conditions, was severely compromised in mice lacking the Atf4 gene. Loss of ATF4 completely prevented endothelial sprouting from embryonic metatarsals, which was restored by addition of recombinant human VEGF protein. In vitro studies revealed that ATF4 promotion of HIF-1? and VEGF expression in osteoblasts was highly dependent upon the presence of hypoxia. ATF4 interacted with HIF-1? in hypoxic osteoblasts, and loss of ATF4 increased HIF-1? ubiquitination and reduced its protein stability without affecting HIF-1? mRNA stability and protein translation. Loss of ATF4 increased the binding of HIF-1? to prolyl hydroxylases, the enzymes that hydroxylate HIF-1a protein and promote its proteasomal degradation via the pVHL pathway. Furthermore, parathyroid hormone-related protein (PTHrP) and receptor activator of NF-?B ligand (RANKL), both well-known activators of osteoclasts, increased release of VEGF from the bone matrix and promoted angiogenesis through the protein kinase C- and ATF4-dependent activation of osteoclast differentiation and bone resorption. Thus, ATF4 is a new key regulator of the HIF/VEGF axis in osteoblasts in response to hypoxia and of VEGF release from bone matrix, two critical steps for bone angiogenesis.
Project description:The von Hippel-Lindau tumor suppressor (pVHL) is a component of an E3 ubiquitin ligase and targets hypoxia-inducible factor-1alpha (HIF-1alpha) for ubiquitylation and degradation under normoxic conditions. pVHL also directly inhibits HIF-1alpha transactivation by recruiting histone deacetylases. Here, we report a novel pVHL-interacting protein that functions as a negative regulator of HIF-1alpha transactivation. This protein, generated from the ZnF197 locus by alternative splicing, contains a Kruppel-associated box (KRAB)-A domain and a SCAN domain, but lacks the 22 C2H2-type zinc fingers present in ZnF197. Therefore, we named this protein pVHL-associated KRAB-A domain-containing protein (VHLaK). We demonstrate that the KRAB-A domain in VHLaK mediates pVHL binding and functions as a transcriptional repression module. The SCAN domain mediates VHLaK homo-oligomerization, which enhances VHLaK repressive activity. pVHL can recruit VHLaK to repress HIF-1alpha transcriptional activity and HIF-1alpha-induced VEGF expression. Finally, we demonstrate that pVHL, VHLaK and KAP1/TIF-1beta can be recruited into a single complex, indicating that KAP1/TIF-1beta may participate in pVHL-mediated transcriptional repression of HIF-1alpha. Our findings provide a novel mechanism for the modulation of HIF-1alpha transactivation by pVHL.
Project description:It is definitively established that mutations in transcription factor HIF-2? are causative of both neuroendocrine tumors (class 1 disease) and polycythemia (class 2 disease). However, the molecular mechanism that underlies this emergent genotype-phenotype relationship has remained unclear. Here, we report the structure of HIF-2? peptide bound to pVHL-elongin B-elongin C (VBC) heterotrimeric complex, which shows topographical demarcation of class 1 and 2 mutations affecting residues predicted, and demonstrated via biophysical analyses, to differentially impact HIF-2?-pVHL interaction interface stability. Concordantly, biochemical experiments showed that class 1 mutations disrupt pVHL affinity to HIF-2? more adversely than class 2 mutations directly or indirectly via impeding PHD2-mediated hydroxylation. These findings suggest that neuroendocrine tumor pathogenesis requires a higher HIF-2? dose than polycythemia, which requires only a mild increase in HIF-2? activity. These biophysical data reveal a structural basis that underlies, and can be used to predict de novo, broad genotype-phenotype correlations in HIF-2?-driven disease.